Antioxidant Activities of Aqueous Extracts of Selected Tropical Plants. Wong S. P. 1 and Leong L. P. 2

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1 Antioxidant Activities of Aqueous Extracts of Selected Tropical Plants Wong S. P. 1 and Leong L. P. 2 Food Science and Technology Programme, c/o Department of Chemistry, National University of Singapore, Singapore , Singapore. Abstract The antioxidant properties of 25 tropical plants were studied using the DPPH free radical scavenging and ferric reducing antioxidant potential assays. Their copper ion chelating activities and total phenolics contents (TPC) were also determined. A strong correlation between TEAC TEAC and TEAC FRAP obtained implied that compounds present in the aqueous extracts capable of donating hydrogen atoms to DPPH were also able to reduce ferric iron via single electron donation. A good correlation of TPC with TEAC DPPH and TEAC FRAP suggested that phenolic compounds in plants except petai, were likely to be the main contributors of antioxidant activities of their aqueous extracts where they could act as HAT and SET antioxidants. The correlation of copper(ii) chelating activity with TPC was poor. Organic acids and some sugars could be sequesters of the copper ions, in addition to phenolics present in the extracts. Introduction Free radical reactions occur both in the human body as well as in food systems. Reactive oxygen and nitrogen radical species (ROS/RNS) are an integral part of normal physiology (Halliwell and Gutteridge, 1989). The over-production of these reactive species due to oxidative stress can cause damage to biomolecules and cause cellular injury and death, which may lead to various chronic diseases such as cancers, cardio- and cerebrovascular diseases. In food systems, antioxidants have been used to prolong the shelf life of foods rich in polyunsaturated fatty acids and their derivatives which are readily oxidised by molecular oxygen via lipid peroxidation. This reaction is a major cause of quality deterioration, nutritional losses, off-flavour development and discolouration in foods. Synthetic antioxidants, such as butylated hydroxytoluene (BHT) have been widely used industrially to control lipid oxidation in foods. However, the use of these synthetic antioxidants has been questioned due to their health risks and toxicity. Hence numerous efforts have been put in to search and identify compounds that can act as suitable antioxidants to replace synthetic ones (Löliger, 1991). In addition, naturally occurring antioxidants, when consumed, can also act as neutraceuticals which can help protect one from oxidative damage in the body. The objectives of this investigation were to determine the total polyphenol contents and characterise the free radical scavenging, iron(iii) ions reducing and copper(ii) ions chelating capabilities of some tropical plants unique to the Southeast Asian region. Results from this preliminary study will help to provide a better understanding of antioxidant properties of these plants and identify plants with high antioxidants for further investigation and development into value-added foods and neutraceuticals. 1 UROPS Student. 2 Supervisor. Tel.: +(65) ; Fax: +(65) address: laipeng@nus.edu.sg (L. P. Leong).

2 Methods and Materials Plant Materials, Chemicals & Sample Preparation All chemicals used were of at least analytical grade obtained from Sigma Chemicals Co. (MO, USA), Acros Organics (NJ, USA), Merck (Darmstadt, Germany) and Aldrich Co. (Milwaukee, WI). Twenty-five plants (listed in Appendix) were obtained fresh from several Singapore wet-markets on separate occasions. Samples were first washed with water to remove all debris and damaged and diseased portions were removed. The leaves of the samples were dried in a convectional oven at 45 o C for 48 hr until there was no change in weight. Dried leaves were ground using a domestic blender and 0.5 g of this material was extracted using 25 ml of deionised water. The mixture was allowed to stand at room temperature for 1 hr in the dark, with occasional agitation. The aqueous extract was obtained by filtering the mixture through Whatman No. 1 filter paper and used for analysis without further treatment. All spectrophotometric work was performed using the Ultraspec 3000 UV/Visible Spectrophotometer (Pharmacia Biotech Ltd., Cambridge CB4 4FJ, UK). Methods DPPH Free Radical Scavenging Assay. Free radical-scavenging activity was determined using the according to the method described by Leong and Shui (2002). The antioxidant capacity of plants was expressed as Trolox Equivalent Antioxidant Capacity (TEAC DPPH ): µmol Trolox per gram of dried plant material. Ferric Reducing Antioxidant Potential (FRAP) Assay. The ability to reduce iron(iii) ions was measured using the method described by Benzie and Strain (1996). The antioxidant capacity of plants was expressed as Trolox Equivalent Antioxidant Capacity (TEAC FRAP ): µmol per gram of dried plant material. Total Phenolics Contents (TPC) Determination. Total phenolics content was determined using the Folin-Ciocalteu s Phenol Reagent and expressed as Gallic Acid Equivalents (GAE): mg gallic acid per gram of dried plant material. Copper(II) Ions Chelation Assay. Copper(II) ions chelating activities (% of Cu 2+ ions chelated) were determined using a modified procedure described by Wettasinghe and Shahidi (2002). Results & Discussion In this study, water was used as the extraction solvent to extract hydrophilic antioxidants from selected tropical plants. For use in foods, plant extracts made with water are nutritionally more relevant and have obvious advantages in relation to certification and safety. Currently, the availability of information on the antioxidant properties of many tropical plants are sporadic and lacking. The antioxidant capacities, total phenolics contents and copper(ii) ions chelating activities were tabulated in Appendix 1. Ranking of the plants according to their activities was not performed as there was significant overlap of the values when the natural variation was considered, although the mean values did show a trend. This has also been encountered in a study of common vegetables conducted by Ou et al. (2002). Free radicals are reactive species with an unpaired electron. Antioxidants are able to reduce free radicals by donating an electron or hydrogen atom to the free radical. In this study, the hydrogen atom transfer (HAT) and the single electron transfer (SET) capabilities were considered separately. The HAT activity of plant extracts was studied using the DPPH free radical and its reaction with a phenolic antioxidant can be written as: DPPH + ArOH DPPH 2 + ArO

3 Antioxidants can also exert their action via SET. The antioxidative action involving SET could be followed using Ferric Reducing Antioxidant Power (FRAP) assay developed by Benzie and Strain (1996). On reaction with an antioxidant capable of donating a single electron, the Fe(III)-TPTZ is reduced to the blue Fe(II)-TPTZ complex which absorbs strongly at 593 nm. A simple SET equation for a polyphenol, in this case, would be: ArOH + [Fe(TPTZ) 2 ] 3+ ArOH + + [Fe(TPTZ) 2 ] 2+ SET has been reported to be highly solvent dependent. The FRAP assay has been criticised for not being representative of conditions in vivo or food systems due to its low ph which may prevent electron transfer. In addition, the usage of iron(iii) ions was too arbitrary as the antioxidant activity of a compound would be totally dependent on its ability to reduce iron Ou et al. (2002). The total phenolics content of the plant extracts was determined using the Folin- Ciocalteu s Phenol reagent. The exact mechanism of the reaction is complex but it is essentially a redox reaction occurring between antioxidants with phosphotungstic and phosphomolybdic acids. Since the reaction is based on redox, the assay would not be specific to just phenolics but to any other substance that could be oxidised by the Folin reagent. This was not surprising as numerous workers has reported the poor specificity of this assay (Singleton et al.,1999; Escarpa and González, 2001). In this study, the removal of the petai data point gave better correlations with the DPPH and FRAP assays (see Table 1). This could be attributed to the presence of substances reactive towards the Folin reagent but not the two other assays. Consequently, a good correlation of TPC with TEAC DPPH and TEAC FRAP obtained in this study implied phenolic compounds in plants except petai, were likely to be the main contributors of antioxidant activities of their aqueous extracts where they could act as HAT and SET antioxidants depending on the reaction conditions used. The extremely strong correlation between TEAC DPPH and TEAC FRAP given in Table 1, deserves detail attention. This could be explained from the basic concept that antioxidants are reducing agents. Antioxidants are compounds capable of donating a single electron or hydrogen atom for reduction. However, this would not be the case for reducing agents as not all reducing agents are antioxidants! In this study, compounds present in the aqueous extracts capable of donating hydrogen atoms to DPPH were also able to reduce ferric iron via single electron donation. The probable reason for the lower TEAC DPPH values of the plants could be due to the presence of other reducing agents not reactive towards DPPH. Antioxidant compounds like polyphenols may be good reducing agents for iron but may not scavenge DPPH due to steric reasons. There might also be reducing agents which are not antioxidants present in the extracts. TEAC FRAP TPC TEAC DPPH TEAC FRAP TPC a b b c c Cu 2+ Chelation b b b Table 1: Correlation Coefficients (R 2 ) for Relationships between Assays a all plants (p<0.01); b all plants (p<0.05); c except Petai (p<0.05)

4 Copper(I) and iron(ii) ions present in vivo and in vitro systems have been shown to be important catalysts for the generation of highly reactive hydroxyl radicals via the Fenton reaction. Metal ions in the lower oxidation state can be produced from the reduction of metal ions in the higher oxidation states by reducing agents such as ascorbic acid and phenolic compounds, thus acting as proxidants. Chelating agents can bind to metal ions, changing their redox potentials rendering the ions catalytically silent. They can also wrap themselves around the ions where they intercept and suppress radicals formed during catalysis from fuelling a chain reaction. The correlations of copper(ii) chelating activity with all other assays were very poor, especially with TPC. This result showed that phenolics, although they have been shown in numerous studies to be relatively good chelators of metal ions, might not be the main chelators of copper ions. In a complex plant matrix, organic acids and some sugars can be sequesters of the copper ions too. The poor correlations of copper(ii) chelating activity with TEAC DPPH and TEAC FRAP, showed that the chelating activities of compounds have little relation with their HAT or SET capabilities, if any. Conclusion Antioxidant compounds present in aqueous plant extracts found to be able to donate hydrogen atoms to DPPH were also able to reduce ferric iron via single electron donation. Phenolic compounds found in plants studied were likely to be the main contributors of antioxidant activities of their aqueous extracts where they could act as HAT and SET antioxidants. Phenolics were not responsible for the copper(ii) ions chelation activities of the extracts. A simple and perfect test that would be able to measure all possible antioxidant mechanisms does not exist and test methods are not immune to interferences present in complex plant extracts. Hence a number of assaying methods are necessary for the characterisation of total antioxidant activities of plant extracts, food and biological samples. One also need to be aware of the possible interferences and limitations of the assay method used so that the possibility of arriving at wrong conclusions would be minimised. References Benzie I. F. F. and Strain J. J. (1996), The Ferric Reducing Ability of Plasma (FRAP) as a Measure of Antioxidant Power : The FRAP Assay, Analytical Biochemistry, 239, Escarpa A. and González M.C. (2001), Approach to the content of total extractable phenolic compounds from different food samples by comparison of chromatographic and spectrophotometric methods, Analytica Chimica Acta, 427, Halliwell B. and Gutteridge J. M. (1989), Free Radicals in Biology and Medicine, New York: Oxford University Press. Leong L. P. and Shui G. (2002), An investigation of antioxidant capacity of fruits in Singapore markets, Food Chemistry, 76, Löliger J. (1991), In Free Radicals and Food Additives (Aruoma O. I. and Halliwell B., eds) pp , Taylor & Francis. Ou B.; Huang, D.; Hampsch-Woodill M.; Flanagan J. A. and Deemer E. K. (2002), Analysis of Antioxidant Activities of Common s Employing Oxygen Radical Absorbance Capacity (ORAC) and Ferric Reducing Antioxidant Power (FRAP) Assays: A Comparative Study, Journal of Agricultural and Food Chemistry, 50, Singleton V. L., Orthofer R. and Lamuela-Raventos R. M. (1999), Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin-Ciocalteu Reagent, Methods in Enzymology, 29, Wettasinghe M. and Shahidi F. (2002), Iron(II) chelation activity of extracts of borage and evening primrose meals, Food Research International, 35,

5 APPENDIX DPPH Radical Scavenging Activity, Iron(III) Ions Reducing Activity, Total Phenolic Contents & Copper(II) Ions Chelation Activity of Tropical Plants used as s &s a Scientific Name Asian Pennywort Centella asiatica Cekur Manis Sauropus androgynus Chinese Boxthorn Lycium chinense Horse Radish Tree Moringa pteriosperma Melinjau Gnetum genom Pucuk Paku Diplazium esculentum Red Amaranthus Amaranthus cruentus L Roselle Hibiscus sabdariffa Sweet Potato Ipomea babatas Tapioca Manihot esculenta L Ulam Raja Cosmos caudatus West Indian Pea Tree Sesbania grandiflora Betel Piper betel Coriander Coriandrum sativum L Curry Tree Murraya koenigii Daun Salam Eugenia polyantha Ketumbar Jawa Eryngium foetidum L Laksa Polygonium odoratum Local Celery Apium graveolens L Mint Mentha arvensis L Petai Parkia speciosa Rue Ruta graveolens L Spring Onion Allium fistulosum Thai Basil Ocimum basilicum L Wild Lime Citrus hystrix Type DPPH Radical Scavenging Activity b (12.80) (19.92) (5.76) (8.91) (6.22) (10.92) (4.68) (7.95) (18.95) (16.60) (36.26) (2.77) (18.58) (7.99) (12.65) (24.39) (5.87) (11.32) (2.62) (33.77) (5.93) (3.90) (1.93) (6.01) (7.59) Iron(III) Ions Reducing Activity c (18.55) (19.29) (12.53) (12.25) (8.04) (7.80) (8.15) (14.14) (32.32) (25.97) (23.33) (6.97) (28.42) (15.03) (15.67) (32.10) (7.05) (13.18) (4.36) (47.22) (13.81) (11.16) (5.21) (24.93) (14.58) Total Phenolics Content d (2.08) (2.59) (1.21) (1.30) (1.77) (1.54) (1.26) (1.80) (2.96) (2.73) (2.58) (1.21) (2.82) (1.82) (2.18) (2.15) 9.16 (0.78) (1.13) (5.42) (3.84) (0.60) 8.11 (0.74) (1.84) Cu 2+ Ions Chelation Activity e (5.68) (3.51) (6.82) (6.68) (13.36) (1.72) (4.02) (5.10) (4.11) (2.48) (3.30) (13.36) (13.84) (6.65) (9.31) (5.67) (5.69) (9.32) (1.71) (14.32) (5.04) (8.39) a Values are the mean of 6 replicates (n = 6). b,c Values expressed as µmol Trolox Equivalents per g dry plant material. d Values expressed as mg Gallic Acid Equivalents per g of dry plant material. e Values expressed as percentage (%) of copper(ii) ions chelated. Values are presented as means (standard deviation). (2.62) (4.04)